采用 Dionex CaboPacTM PA10 BioLCTM Analyical 2 mm × 250 mm 色谱柱，配以保护柱(Dionex CaboPacTM PA10BioLCTM Guard 2 mm × 50 mm);以100 mmol·L-1 氢氧化钠溶液为淋洗液;流速为0。25 mL·min-1;样品盘温度:35 ℃。采用脉冲安培检测器，波形为:Gold CWE，Ag-AgCl RE，Carbo，Quad。以8个浓度的糖原底物(0。31、1。25、2。5、5、10、20、30和40mg·mL-1)培养样品。在5个不同时间点(T0～T4)时测定葡萄糖浓度。T1～T4的葡萄糖浓度减去T0时的葡萄糖浓度，在不同糖原底物浓度下，计算反应速率。利用米氏方程，绘制这些反应速率与底物浓度的曲线。以Vmax(nmol·mg-1·min-1)和Km(mg·mL-1)表示动力学参数。葡萄糖标准曲线(线性范围:1。25～500 μmol·L-1)，葡萄糖标准曲线的系数R2(n=6)的RSD为0。1％，标准曲线斜率RSD为2。2％;定量限平均值为0。14 μmol·L-1，检测限平均值为0。05 μmol·L-1;供试品重复3次单独实验Km的RSD为4。4％，Vmax的RSD为4。6％，方法耐用性良好。建立离子色谱法在线自动化测定注射用阿糖苷酶α糖原水解动力学的方法，该方法精密度、重复性和耐用性良好，可以用于注射用阿糖苷酶α糖原水解动力学的检测，并且对重组酶替代疗法酶类的糖原动力学评价具有参考价值。
Establishment of a method for determining the key parameters of hydrolysis kinetics of acid α-glucosidase for injection by ion chromatography
The Dionex CaboPacTM PA 10 BioLCTM Analyical 2 mm × 250 mm column was used with a protective column(Dionex CaboPac™ PA10 BioLCTM Guard 2 mm × 50 mm).100 mmol·L-1 sodium hydroxide solution was used as eluent;the flow rate was 0.25 mL·min-1.Sample tray temperature:35 ℃.The pulse amperometric detector was adopted,and the waveform was Gold CWE,Ag-AgCl RE,Carbo,Quad.The samples were cultured with 8 concentrations of glycogen substrates(0.31,1.25,2.5,5,10,20,30,and 40 mg·mL-1).D-Glucose concentrations were measured at 5 different time points(T0,T1,T2,T3 and T4).The glucose concentration from T1 to T4 minus the glucose concentration at T0.The reaction rate was calculated at different glycogen substrate concentrations.These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation.The kinetic parameters were expressed as Vmax(nmol·mg-1·min-1)and Km(mg·mL-1).The RSD of glucose standard curve R2(n=6,linear range:1.25-500 μmol·L-1)was 0.1％and the RSD(n=6)of the slope of the standard curve was 2.2％.The mean limit of quantitation was 0.14 μmol·L-1,and the mean limit of detection was 0.05 μmol·L-1.The RSD of Km and Vmax were 4.4％and 4.6％respectively in three separate experiments.The durability of the method was good.The method was developed for the on-line automatic determination of the hydrolysis kinetics of acidα-glucosidase(GAA)for injection by ion chromatography.The method has good precision,repeatability and durability,and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection,and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.
acid α-glucosidase for injectionion chromatographypulse amperometric detectorMichaelis-Menten equationenzyme kineticson-line automation
万方数据
维普
